Viruses and many bacteria must invade a cell to replicate, and cell entry is a step usefully accessible to inhibitors. Studies of pathogen entry have advanced our understanding of basic cell biological processes and led to discovery of new molecular mechanisms, while also providing opportunities to consider novel strategies for intervention. During the last decade, approaches using biochemistry, genetics, and real-time imaging have led to identification of a number of alternative modes of pathogen uptake, with sometimes subtle, yet critical differences in the way particular cellular structures are deployed. Direct fluorescence imaging of individual internalization events, combined when appropriate with pharmacological or genetic perturbation, resolves ambiguities associated with ensemble measurements, and we rely on its use throughout this proposal. This project focuses on understanding the role of multiple modes of clathrin-mediated endocytosis for viral entry and how they relate to the destination of endocytosed virus, information that is relevant to strategies for inhibiting entry, to analysis of cell tropism and different outcomes for infection of different tissues, and to relating information from entry of pseudotyped viruses with heterologous envelope proteins to entry of authentic virions. This project also aims to understand the role of the clathrin endocytic machinery in infection by invasive and adherent bacterial pathogens.